The cyclic 3, '5'-adenosine monophosphate (cyclic AMP)-dependent protein kinase in skeletal muscle appears to be a hormonally regulated enzyme which catalyzes the transfer of the terminal phosphoryl group of MgATP2- to specific phosphoryl accepting amino acid residues of at least two enzymes (phosphorylase kinase and glycogen synthase) and one protein (protein inhibitor I of protein phosphatase III). These enzymes/proteins are, if not controlled, potentially involved in the futile cycle of important pathways of glycogen metabolism in skeletal muscle. It is, therefore, important to investigate the interactions between cyclic AMP-dependent protein kinase and its enzyme/protein substrate as well as its modulator (cyclic AMP). The long-range objectives of this proposal are (1) to answer the question of what chemical groups on the active center determine the substrate specificity; (2) to determine the structural features of the protein/enzyme substrates which characterize the specific protein kinase - substrate interaction; (3) to investigate the mechanism of cyclic AMP in the activation of cyclic AMP-dependent protein kinase, and (4) to examine the possible involvement of disulfide bridges in the inhibitory action of dimeric regulatory subunity on the holoenzyme. The study will utilize homogeneous cyclic AMP-dependent protein kinase, Type I, and its subunits isolated from rabbit skeletal muscle. The detail of the proposed research will be established by systematic studies involving chemical group modifications, gel and paper electrophoresis, molecular sieve chromatography, chemical cross-linking, steady-state initial kinetics, kinetics with inhibitors.